We show that a knot in $S^3$ with an infinite number of distinct incompressible Seifert surfaces contains a closed incompressible surface in its complement.

The literature suggests that glare sensation may be influenced by visual task difficulty. Previous research by the authors provided reasons to infer that the perceived level of visual discomfort may vary with time of day and be affected by temporal and personal factors. The study presented here explores the postulated relationships between visual task difficulty, temporal variables, and glare response as the day progresses. Under controlled laboratory conditions, twenty subjects were exposed to a constant artificial source luminance at four times of day and gave glare sensation votes while completing twelve visual tasks of various difficulties. Self-assessments of temporal variables (fatigue, food intake, caffeine ingestion, mood, previous daylight exposure and sky condition) were provided by test subjects together with their glare judgements. Statistical analysis of responses confirmed that the time interval between test sessions showed a direct relationship to the increased tolerance to artificial source luminance along the day. The temporal variation of glare response wasfound to be influenced by the difficulty in extracting information from the visual stimulus. Moreover, statistically significant and substantive evidence was detected of a direct effect of fatigue and caffeine ingestion, and an inverse influence of food intake, on reported glare sensation. Consideration of inferential results from all test sessions led to hypothesise that some temporal variables may interact with each other and significantly affect the variation of glare response at different times of day.

Thermochromic windows have been widely studied as a technology that can potentially offer increases in energy conservation and provide a desirable luminous environment inside buildings. However, there has been little attention placed on how the tinted states of thermochromic glazing influence occupant behaviour and visual perception. An experiment under controlled conditions was designed to test the influence of different thermochromic tint states on human response. By using a controllable artificial window, five typical luminous conditions were set up, including clear (no tint) and two different levels of blue and bronze tint states, respectively, which produced different room colour temperatures. Thirty-one subjects were recruited who completed three visual tasks, including a visual acuity and a colour naming tasks using the coloured Landolt ring chart and a sustained attention test using the d2 test. Subjective assessments were also collected using questionnaires. Statistical analyses showed the across the thermochromic window conditions, no significant differences in performance were found for the visual acuity and d2 tests. However, there was a significant effect for the colour naming task from the Landolt ring test. Under blue tint conditions, subjects reported higher alertness and produced fewer errors. More natural and acceptable lighting conditions were found under the bronze-tinted conditions. Therefore, when developing innovative thermochromic windows applied in buildings, it is also important to cater for the visual requirements of the occupants in the space, not only energy efficiency goals.

While performing several functions, adherent cells deform their surrounding substrate via stable adhesions that connect the intracellular cytoskeleton to the extracellular matrix. The traction forces that deform the substrate are studied in mechanotrasduction because they are affected by the mechanics of the extracellular milieu. We review the development and application of two methods widely used to measure traction forces generated by cells on 2D substrates: (i) traction force microscopy with polyacrylamide hydrogels and (ii) calculation of traction forces with arrays of deformable microposts. Measuring forces with these methods relies on measuring substrate displacements and converting them into forces. We describe approaches to determine force from displacements and elaborate on the necessary experimental conditions for this type of analysis. We emphasize device fabrication, mechanical calibration of substrates and covalent attachment of extracellular matrix proteins to substrates as key features in the design of experiments to measure cell traction forces with polyacrylamide hydrogels or microposts. We also report the challenges and achievements in integrating these methods with platforms for the mechanical stimulation of adherent cells. The approaches described here will enable new studies to understand cell mechanical outputs as a function of mechanical inputs and advance the understanding of mechanotransduction mechanisms.

Introduction

Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization.

Methods

We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (3D/3D), 3D microwells with 2D adhesive areas at the bottom only (3D/2D), and flat 2D gels with 2D patterned adhesive areas (2D/2D). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape.

Results

We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment.

Conclusions

Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

Introduction

Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs 3D geometries on single-cell F-actin and nuclear organization.

Methods

We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment ( 3D/3D ), 3D microwells with 2D adhesive areas at the bottom only ( 3D/2D ), and flat 2D gels with 2D patterned adhesive areas ( 2D/2D ). We measured geometric swelling and Young’s modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape.

Results

We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young’s modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment.

Conclusions

Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.

While performing several functions, adherent cells deform their surrounding substrate via stable adhesions that connect the intracellular cytoskeleton to the extracellular matrix. The traction forces that deform the substrate are studied in mechanotrasduction because they are affected by the mechanics of the extracellular milieu. We review the development and application of two methods widely used to measure traction forces generated by cells on 2D substrates: (i) traction force microscopy with polyacrylamide hydrogels and (ii) calculation of traction forces with arrays of deformable microposts. Measuring forces with these methods relies on measuring substrate displacements and converting them into forces. We describe approaches to determine force from displacements and elaborate on the necessary experimental conditions for this type of analysis. We emphasize device fabrication, mechanical calibration of substrates and covalent attachment of extracellular matrix proteins to substrates as key features in the design of experiments to measure cell traction forces with polyacrylamide hydrogels or microposts. We also report the challenges and achievements in integrating these methods with platforms for the mechanical stimulation of adherent cells. The approaches described here will enable new studies to understand cell mechanical outputs as a function of mechanical inputs and advance the understanding of mechanotransduction mechanisms.

Human-induced pluripotent stem cell-derived cardiomyocytes are a potentially unlimited cell source and promising patient-specific in vitro model of cardiac diseases. Yet, these cells are limited by immaturity and population heterogeneity. Current in vitro studies aiming at better understanding of the mechanical and chemical cues in the microenvironment that drive cellular maturation involve deformable materials and precise manipulation of the microenvironment with, for example, micropatterns. Such microenvironment manipulation most often involves microfabrication protocols which are time-consuming, require cleanroom facilities and photolithography expertise. Here, we present a method to increase the scale of the fabrication pipeline, thereby enabling large-batch generation of shelf-stable microenvironment protein templates on glass chips. This decreases fabrication time and allows for more flexibility in the subsequent steps, for example, in tuning the material properties and the selection of extracellular matrix or cell proteins. Further, the fabrication of deformable hydrogels has been optimized for compatibility with these templates, in addition to the templates being able to be used to acquire protein patterns directly on the glass chips. With our approach, we have successfully controlled the shapes of cardiomyocytes seeded on Matrigel-patterned hydrogels.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are powerful in vitro models to study the mechanisms underlying cardiomyopathies and cardiotoxicity. Quantification of the contractile function in single hiPSC-CMs at high-throughput and over time is essential to disentangle how cellular mechanisms affect heart function. Here, we present CONTRAX, an open-access, versatile, and streamlined pipeline for quantitative tracking of the contractile dynamics of single hiPSC-CMs over time. Three software modules enable: parameter-based identification of single hiPSC-CMs; automated video acquisition of >200 cells/hour; and contractility measurements via traction force microscopy. We analyze >4,500 hiPSC-CMs over time in the same cells under orthogonal conditions of culture media and substrate stiffnesses; +/- drug treatment; +/- cardiac mutations. Using undirected clustering, we reveal converging maturation patterns, quantifiable drug response to Mavacamten and significant deficiencies in hiPSC-CMs with disease mutations. CONTRAX empowers researchers with a potent quantitative approach to develop cardiac therapies.